8    ◾    Bioinformatics

nucleotide. The base call is based on the intensity of the signals during chain synthesis and

labeled nucleotide incorporation.

The DNA library preparation, as described above, includes the DNA fragmentation,

DNA end repair, and adaptor ligation. Sequencing is carried out on the solid surface of a

flow cell divided into lanes. On the surface, there are two types of oligonucleotides that

complement the anchor sequence in the adaptors attached to the DNA template. When the

DNA fragments are added to the cell flow, the anchor sequences in the fragments anneal

to (complement) the surface oligonucleotides forming bridges. The oligos also act as prim-

ers that initiate the synthesis of complementary strands generating clusters of the DNA

fragments. The sequencing step begins by denaturing the DNA strands and adding fluo-

rescently labeled nucleotides. Only one nucleotide is incorporated in the DNA template at

a time. After the addition of each nucleotide, the clusters are excited by a light source and

a signal is emitted. The signal intensity determines the base call.

1.2.3  Third-Generation Sequencing

The third-generation sequencing (TGS) is relatively new sequencing technology developed

as a result of the need for long reads to improve the accuracy and resolution of sequencing

FIGURE 1.4  Illumina sequencing.